Inhibition of p38 mitogen-activated protein kinase activation in the rostral anterior cingulate cortex attenuates pain-related negative emotion in rats

doi:10.1016/j.brainresbull.2014.06.005

Brain Research Bulletin

Volume 107, August 2014, Pages 79-88

https://doi.org/10.1016/j.brainresbull.2014.06.005 Get rights and content

Highlights

•
Peripheral noxious stimuli upregulate p-p38 MAPK and pMKK3/6 levels.
•
Activation of p38 MAPK is necessary for pain-related negative emotion.
•
p-p38 MAPK exists in both neurons and microglia in the bilateral ACC.

Abstract

The emotional components of pain are far less studied than the sensory components. Previous studies have indicated that the rostral anterior cingulate cortex (rACC) is implicated in the affective response to noxious stimuli. Activation of p38 mitogen-activated protein kinase (MAPK) in the spinal cord has been documented to play an important role in diverse kinds of pathological pain states. We used formalin-induced conditioned place aversion (F-CPA) in rats, an animal model believed to reflect the emotional response to pain, to investigate the involvement of p38 MAPK in the rACC after the induction of affective pain. Intraplantar formalin injection produced a significant activation of p38 MAPK, as well as mitogen-activated kinase kinase (MKK) 3 and MKK6, its upstream activators, in the bilateral rACC. p38 MAPK was elevated in both NeuN-positive neurons and Iba1-positive microglia in the rACC, but not GFAP-positive cells. Blocking p38 MAPK activation in the bilateral rACC using its specific inhibitor SB203580 or SB239063 dose-dependently suppressed the formation of F-CPA. Inhibiting p38 MAPK activation did not affect formalin-induced two-phase spontaneous nociceptive response and low intensity electric foot-shock induced CPA. The present study demonstrated that p38 MAPK signaling pathway in the rACC contributes to pain-related negative emotion. Thus, a new pharmacological strategy targeted at the p38 MAPK cascade may be useful in treating pain-related emotional disorders.

Introduction

Pain experience incorporates both sensory and affective dimensions. The former describes the location, intensity and quality of the stimuli. The latter describes unpleasantness, aversion or fear to the pain experience, thereby producing the desire to terminate or escape from the noxious stimuli. Clinical observations indicate that the patients with chronic pain suffer from as much emotional disturbance as pain sensation (Crombez et al., 1999). In response to physiological arousal and hypersensitivity to pain, patients experience fear, anxiety, anger, depression and even a suicidal tendency. These negative affective states in turn enhance pain perception. Recently, pain-related emotion has received more attention.

Accumulating evidence from morphological, electrophysiological, neuroimaging, and behavioral studies as well as early clinical observation indicates that the anterior cingulate cortex (ACC) is related to pain-related emotion, cognition, memory and anticipation (Foltz and White, 1968, Gao et al., 2004, Johansen et al., 2001, Koyama et al., 1998, Kung et al., 2003, Price, 2000, Rainville et al., 1997). Further studies from other groups and our laboratory demonstrate that activation of N-methyl-d-aspartic acid (NMDA) receptors (NMDAR) and the protein kinase A (PKA)–extracellular signal-regulated kinase (ERK)–cyclic adenosine monophosphate response element binding protein (CREB) signaling pathway in the rACC is required for the induction of pain-related negative emotion (Cao et al., 2009; Johansen and Fields, 2004; Ren et al., 2006). This signaling pathway is also strongly implicated in pain hyperalgesia in the spinal cord, suggesting pain sensation and pain emotion might share similar signaling pathways in different central regions.

The mitogen-activated protein kinases (MAPKs) are a family of signaling molecules that transduce extracellular stimuli into intracellular responses in a wide variety of circumstances. Activation of p38 MAPK requires dual phosphorylation of the threonine 180 (Thr180) and tyrosine 182 (Tyr182) residues within the conserved TGY motif (Widmann et al., 1999). Studies demonstrate that the activation of p38 MAPK cascade in the spinal cord following peripheral noxious stimulation contributes to both short-term and long-term pain hypersensitivity (Crown et al., 2008, Jin et al., 2003, Ito et al., 2007, Sukhtankar et al., 2011, Schafers et al., 2003, Svensson et al., 2003, Sweitzer et al., 2004, Tan et al., 2012, Zhuang et al., 2007). While evidence shows that the ACC is involved in pain emotion, whether the p38 MAPK cascade in the ACC contributes to pain-related aversion remains unknown. The overall goal of our study is to investigate the activation of p38 MAPK in the ACC following peripheral noxious stimulation and its contribution to persistent pain-induced negative emotion.

Section snippets

Animals and reagents

Experiments were performed on adult (weighting 220–250 g) male Sprague-Dawley rats. Animals were obtained from Experimental Animal Center of the Chinese Academy of Science (CAS) and were on a 12:12 light–dark cycle with a room temperature of 23 ± 10 °C, and received food and water ad libitum. The animals were given a period of 3 days to adjust to the new surroundings before experimental manipulations. All experiments were carried out in according with the guidelines of the International Association

Intraplantar formalin injection activates p38/MAPK and its upstream activators in the rACC

Intraplantar (i.pl.) injection of formalin in the hindpaw of rat elicited characteristic biphasic nociceptive agitation behavior including lifting, licking, shaking and biting. The first phase lasted for about 5 min and the second phase lasted for about 40 min. The level of phospho-p38 (p-p38) MAPK in the bilateral ACC after formalin injection was firstly examined by Western blot. As shown in Fig. 1, formalin injection induced a robust upregulation of p-p38 MAPK in the bilateral ACC (one-way

Discussion

Activation of p38 MAPK in the spinal cord is required for nociceptive pain sensitization in both inflammatory and neuropathic pain models (Jin et al., 2003, Ito et al., 2007, Schafers et al., 2003, Svensson et al., 2003, Tsuda et al., 2004; Zhuang et al., 2007). The present study has further shown that intraplantar formalin injection also activates p38 MAPK in the rACC neurons and microglial, and this activation is critical for the induction of affective pain.

Formalin subcutaneously injection

Role of funding source

This work was supported by National Natural Science Fund of China (NSFC 30900444, 31371123, 31121061, and 31271183), China Postdoctoral Science Foundation (2014M551317) and the International Postdoctoral Exchange Fellowship Program 2013 (201355).

Conflict of interest

The authors declare that they have no duality or conflict of interest.

Acknowledgement

These authors wish to thank Dr. Sarah Wardlaw for her helpful criticism and linguistic revision of the manuscript.

References (47)

H. Cao et al.
Spinal glial activation contributes to pathological pain states
Neurosci. Biobehav. Rev.
(2008)
G. Crombez et al.
Pain-related fear is more disabling than pain itself: evidence on the role of pain-related fear in chronic back pain disability
Pain
(1999)
E.D. Crown et al.
Activation of p38 MAP kinase is involved in central neuropathic pain following spinal cord injury
Exp. Neurol.
(2008)
J.A. DeLeo et al.
The role of neuroinflmmation and neuroimmune activation in persistent pain
Pain
(2001)
Y.J. Gao et al.
Contributions of the anterior cingulate cortex and amygdala to pain- and fear-conditioned place avoidance in rats
Pain
(2004)
H. Imbe et al.
Effects of peripheral inflammation on activation of p38 mitogen-activated protein kinase in the rostral ventromedial medulla
Brain Res.
(2007)
R.R. Ji et al.
Central sensitization and LTP: do pain and memory share similar mechanisms?
Trends Neurosci.
(2003)
G. Krapivinsky et al.
SynGAP–MUPP1–CaMKII synaptic complexes regulate p38 MAP kinase activity and NMDA receptor-dependent synaptic AMPA receptor potentiation
Neuron
(2004)
J.C. Kung et al.
Contribution of the anterior cingulate cortex to laser-pain conditioning in rats
Brain Res.
(2003)
L.G. Lei et al.
NMDA receptors in the anterior cingulate cortex mediate pain-related aversion
Exp. Neurol.
(2004)

T.T. Li et al.

NMDA NR2A and NR2B receptors in the rostral anterior cingulated cortex contribute to pain-related aversion in male rats

Pain

(2009)

Y. Lu et al.

Pain-related aversion induces astrocytic reaction and proinflammatory cytokine expression in the anterior cingulate cortex in rats

Brain Res Bull

(2011)

K. Mizukoshi et al.

Activation of p38 mitogen-activated protein kinase in the dorsal root ganglion contributes to pain hypersensitivity after plantar incision

Neuroscience

(2013)

L.S. Sorkin et al.

MKK3, an upstream activator of p38, contributes to formalin phase 2 and late allodynia in mice

Neuroscience

(2009)

S.M. Sweitzer et al.

Peripheral and central p38 MAPK mediates capsaicin-induced hyperalgesia

Pain

(2004)

Y.H. Tan et al.

Activation of Src family kinases in spinal microglia contributes to formalin-induced persistent pain state through p38 pathway

J. Pain

(2012)

L.R. Watkins et al.

Glial activation: a driving force for pathological pain

Trends Neurosci.

(2001)

E.A. Waxman et al.

N-methyl-d-aspartate receptor subtype mediated bidirectional control of p38 mitogen-activated protein kinase

J. Biol. Chem.

(2005)

J.J. Zhu et al.

Ras and Rap control AMPA receptor trafficking during synaptic plasticity

Cell

(2002)

Z.Y. Zhuang et al.

Role of the CX3CR1/p38 MAPK pathway in spinal microglia for the development of neuropathic pain following nerve injury-induced cleavage of fractalkine

Brain Behav. Immun.

(2007)

H. Cao et al.

Activation of extracellular signal-regulated kinase in the anterior cingulate cortex contributes to the induction and expression of affective pain

J. Neurosci.

(2009)

E. Foltz et al.

The role of rostral cingulumotomy in “pain” relief

Int. J. Neurol.

(1968)

U.K. Hanisch

Microglia as a source and target of cytokines

Glia

(2002)

Cited by (15)

Janus effect of the anterior cingulate cortex: Pain and emotion
2023, Neuroscience and Biobehavioral Reviews
Over the past 20 years, clinical and preclinical studies point to the anterior cingulate cortex (ACC) as a site of interest for several neurological and psychiatric conditions. The ACC plays a critical role in emotion, autonomic regulation, pain processing, attention, memory and decision making. An increasing number of studies have demonstrated the involvement of the ACC in the emotional component of pain and its comorbidity with emotional disorders such as anxiety and depression. Thanks to the development of animal models combined with state-of-the-art technologies, we now have a better mechanistic understanding of the functions of the ACC. Hence, the primary aim of this review is to compile the most recent preclinical studies on the role of ACC in the emotional component and consequences of chronic pain. Herein, we thus thoroughly describe the pain-induced electrophysiological, molecular and anatomical alterations in the ACC and in its related circuits. Finally, we discuss the next steps that are needed to strengthen our understanding of the involvement of the ACC in emotional and pain processing.
p38 mitogen-activated protein kinase and pain
2020, Life Sciences
Citation Excerpt :
Meanwhile, the descending pain modulatory system influences nociceptive input at different levels [59]. p38 MAPK phosphorylation can be observed in the related structures of nociceptive pathways that include TG, DRG, spinal cord, cuneate nucleus [60], Vc [22], prelimbic cortex [19], red nucleus [61], rostral anterior cingulate cortex [62], rostral ventromedial medulla [50,55], spinothalamic tract [63], medullary dorsal horn [38,39], dorsal column nucleus [40] and periaqueductal gray [41]. As mentioned above, p38 MAPK can be activated in different pain states and plays an important role in the development and/or maintenance of pain.
Inflammatory and neuropathic pain is initiated by tissue inflammation and nerve injury, respectively. Both are characterized by increased activity in the peripheral and central nervous system, where multiple inflammatory cytokines and other active molecules activate different signaling pathways that involve in the development and/or maintenance of pain. P38 mitogen-activated protein kinase (MAPK) is one member of the MAPK family, which is activated in neurons and glia and contributes importantly to inflammatory and neuropathic pain. The aim of this review is to summarize the latest advances made about the implication of p38 MAPK signaling cascade in pain. It can deepen our understanding of the molecular mechanisms of pain and may help to offer new targets for pain treatment.
Cholinergic/opioid interaction in anterior cingulate cortex reduces the nociceptive response of vocalization in guinea pigs
2017, Brain Research
Citation Excerpt :
On the other hand, GABAergic, opioidergic and dopaminergic stimulation of ACC neurons inhibits the acquisition of aversive memory secondary to nociceptive stimulation (López-Avila et al., 2004; LaGraize et al., 2006; LaGraize and Fuchs, 2007; Pezze et al., 2016). Avoidance conditioning paradigms with nociceptive stimuli have been used to investigate the effects of the modulation of the ACC on the affective-motivational component of nociception (LaGraize et al., 2004; Pellicer et al., 2007; Yan et al., 2012; Cao et al., 2014; Fuchs et al., 2014; Lu et al., 2015). However, other studies have demonstrated that nociceptive motor behaviors and/or reflexes are modulated by electrical and pharmacological manipulation of the ACC (Fuchs et al., 1996; Lee et al., 1999; Calejesan et al., 2000; Zhang et al., 2005; Yi et al., 2011).
The anterior cingulate cortex (ACC) is crucial in the modulation of the sensory, affective and cognitive aspects of nociceptive processing. Also, it participates in the planning and execution of behavioral responses evoked by nociceptive stimuli via descending projections to the brainstem. In laboratory animals nociceptive experimental tests evaluate behavioral responses that preferentially express the sensory-discriminative or affective-motivational component of pain. The objective of this study was to investigate the participation of opioid and cholinergic neurotransmission in the ACC on different nociceptive responses in guinea pigs. We used nociceptive tests of formalin and vocalization evoked by peripheral noxious stimuli (electric shock) to evaluate the behavioral expression of the sensory-discriminative and affective motivational components, respectively. We verified that the microinjection of morphine (4.4 nmol) in the ACC of guinea pigs promotes antinociception in the two experimental tests investigated. This effect is blocked by prior microinjection of naloxone (2.7 nmol). On the other hand, the microinjection of carbachol (2.7 nmol) in the ACC induces antinociception only in the vocalization test. This effect was prevented by prior microinjection of atropine (0.7 nmol) and naloxone (2.7 nmol). In fact, the blockade of µ-opioids receptors with naloxone in ACC prevented the antinociceptive effect of carbachol in the vocalization test. Accordingly, we suggest that the antinociception promoted by carbachol was mediated by the activation of muscarinic receptors on local ACC opioid interneurons. The release of endogenous opioids seems to inhibited the expression of the behavioral response of vocalization. Therefore, we verified that the antinociceptive effect of morphine microinjection in ACC is broader and more robust than that promoted by carbachol.
Role of microglia in mechanical allodynia in the anterior cingulate cortex
2017, Journal of Pharmacological Sciences
Citation Excerpt :
In the ACC, role of activated astrocyte in negative emotion has been reported.15 Moreover, p38 mitogen-activated protein kinase that mainly expressed in microglia modulates pain-related negative emotion.16 Although direct evidence for the role of ACC astrocyte and microglia in sensory function has not been reported, pharmacological inhibition of astrocyte and microglia by gastrodin isolated from Chinese herb into the ACC attenuated the inflammation-induced spontaneous pain.17
Plastic changes that increase nociceptive transmission are observed in several brain regions under conditions of chronic pain. Synaptic plasticity in the anterior cingulate cortex (ACC) is particularly associated with neuropathic pain. Glial cells are considered candidates for the modulation of neural plastic changes in the central nervous system. In this study, we aimed to investigate the role of ACC glial cells in the development of neuropathic pain. First, we examined the expression of glial cells in the ACC of nerve-ligated mice. The expression of astrocytes and microglia was increased in the ACC of nerve-ligated mice, which was reversed by intracerebroventricular (i.c.v) treatment with the microglia inhibitor minocycline. Then, we examined the effect of minocycline on mechanical allodynia in nerve-ligated mice. I.c.v. and intra-ACC treatment with minocycline partially inhibited mechanical allodynia in the nerve-ligated mice. The expression of phosphorylated alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR1 subunit at Ser831, but not at Ser845, was increased in the ACC of the nerve-ligated mice compared to sham-operated mice, which was attenuated by minocycline administration. These results suggest that the activation of microglia in the ACC is involved in the development of hyperalgesia in mice with neuropathic pain.
Calmodulin inhibition regulates morphological and functional changes related to the actin cytoskeleton in pure microglial cells
2016, Brain Research Bulletin
Citation Excerpt :
LPS activation renders microglia ameboid, induces several pro- and anti-inflammatory signaling molecules (Lim et al., 2015; Zhu et al., 2014) and neurotoxic substances through binding to the CD14/MD-2/Toll-like receptor 4-complex (Fricker et al., 2012; Tokes et al., 2011), and gives rise, among others, to cell spreading by interfering with the organization of the actin cytoskeleton through the alteration of integrin clustering (Abram and Lowell, 2009). Microglia activation was shown to involve the signaling pathways nuclear factor κB and p38 mitogen-activated protein kinase (Bachstetter et al., 2011; Cao et al., 2014; Kaushal et al., 2007). It must be noted, however, that the activation of microglial cells by LPS is not proliferative (Suzumura et al., 1991).
The roles of calmodulin (CaM), a multifunctional intracellular calcium receptor protein, as concerns selected morphological and functional characteristics of pure microglial cells derived from mixed primary cultures from embryonal forebrains of rats, were investigated through use of the CaM antagonists calmidazolium (CALMID) and trifluoperazine (TFP). The intracellular localization of the CaM protein relative to phalloidin, a bicyclic heptapeptide that binds only to filamentous actin, and the ionized calcium-binding adaptor molecule 1 (Iba1), a microglia-specific actin-binding protein, was determined by immunocytochemistry, with quantitative analysis by immunoblotting. In unchallenged and untreated (control) microglia, high concentrations of CaM protein were found mainly perinuclearly in ameboid microglia, while the cell cortex had a smaller CaM content that diminished progressively deeper into the branches in the ramified microglia. The amounts and intracellular distributions of both Iba1 and CaM proteins were altered after lipopolysaccharide (LPS) challenge in activated microglia. CALMID and TFP exerted different, sometimes opposing, effects on many morphological, cytoskeletal and functional characteristics of the microglial cells. They affected the CaM and Iba1 protein expressions and their intracellular localizations differently, inhibited cell proliferation, viability and fluid-phase phagocytosis to different degrees both in unchallenged and in LPS-treated (immunologically challenged) cells, and differentially affected the reorganization of the actin cytoskeleton in the microglial cell cortex, influencing lamellipodia, filopodia and podosome formation. In summary, these CaM antagonists altered different aspects of filamentous actin-based cell morphology and related functions with variable efficacy, which could be important in deciphering the roles of CaM in regulating microglial functions in health and disease.
A new potent analgesic agent with reduced liability to produce morphine tolerance
2015, Brain Research Bulletin
Citation Excerpt :
Therefore one of the objectives of the present work was to determine the capability of 14-O-MeM6SU to produce analgesic tolerance in mice chronically treated with morphine or 14-O-MeM6SU. Prefrontal cortical neurons beside their role in the emotional components of pain are also critically involved in withdrawal, reward and tolerance development as indicated by in in vivo and in vitro studies (Cao et al., 2014; Wu et al., 2013). Thus we have also measured the effect of 14-O-MeM6SU on spontaneous excitatory postsynaptic currents (sEPSCs) in layer V pyramidal cells of rat prefrontal cortical slices, compared to morphine to clarify how the new compound may affect glutamatergic transmission.
The therapeutic use of opioids is limited by the development of tolerance to the analgesic effect and the cellular and molecular mechanisms underlying this phenomenon are still not completely understood. For this reason the search for new analgesic derivatives, endowed with lower tolerance, is always an active field. The newly synthesized 14-O-Methylmorphine-6-sulfate (14-O-MeM6SU) shows high efficacy in in vitro assays and a strong analgesic action in the rat tail flick test. The aim of present work was to investigate: the analgesic effect of 14-O-MeM6SU in mouse tail-flick test; the tolerance to analgesic effect of 14-O-MeM6SU compared to morphine in mice, the effects of test compounds on glutamatergic neurotransmission by measuring spontaneous excitatory postsynaptic currents (sEPSCs) of layer V pyramidal cells from rat prefrontal cortices; and the effect of acute and chronic 14-O-MeM6SU treatments on opioid receptor gene expression in SH-SY5Y neuroblastoma cells expressing μ-opioid (MOP) and nociceptin/opioid receptor-like 1 (NOP) receptors.
14-O-MeM6SU was 17 times more potent than morphine in analgesia and had long duration of action in analgesic dose equipotent to morphine. Mice were treated subcutaneously (s.c.) either with 200 μmol/kg morphine or with 14-O-MeM6SU (12 μmol/kg) twice daily for three days. The magnitude of tolerance or cross-tolerance indicated by the shift in antinociceptive ED₅₀ measured was greater for morphine compared to 14-O-MeM6SU. Subsequent to behavioral testing, patch-clamp experiments in layer V pyramidal neurons of rat prefrontal cortical slices in the presence of bicuculline were performed. Both 14-O-MeM6SU (0.1 μM) and morphine (1 μM) decreased the frequency of sEPSCs, indicating reduction of glutamate release. The effect of the novel compound was reversed by the opioid receptor antagonist naloxone, indicating an opioid mediated action. In contrast, the amplitude was not affected. Finally, gene expression data showed a dose dependent down-regulation of MOP receptor after 24 h and 48 h exposure to 14-O-MeM6SU. Interestingly, no changes were detected for NOP receptor gene expression. The specific lack of this effect could be related to the lower tolerance development to analgesic effect of 14-O-MeM6SU. Furthermore, 14-O-MeM6SU displayed high intrinsic efficacy possibly an important factor in the observed effects. Further, the observed inhibition of glutamatergic signaling might be attributed also to the reduction of opioid tolerance. Based on our results the development of a new clinically important, safe analgesic agent might be possible.

View all citing articles on Scopus

View full text

Research reportInhibition of p38 mitogen-activated protein kinase activation in the rostral anterior cingulate cortex attenuates pain-related negative emotion in rats

Highlights

Abstract

Introduction

Section snippets

Animals and reagents

Intraplantar formalin injection activates p38/MAPK and its upstream activators in the rACC

Discussion

Role of funding source

Conflict of interest

Acknowledgement

Neurosci. Biobehav. Rev.

Pain

Exp. Neurol.

Pain

Pain

Brain Res.

Trends Neurosci.

Neuron

Brain Res.

Exp. Neurol.

Pain

Brain Res Bull

Neuroscience

Neuroscience

Pain

J. Pain

Trends Neurosci.

J. Biol. Chem.

Cell

Brain Behav. Immun.

Activation of extracellular signal-regulated kinase in the anterior cingulate cortex contributes to the induction and expression of affective pain

J. Neurosci.

The role of rostral cingulumotomy in “pain” relief

Int. J. Neurol.

Microglia as a source and target of cytokines

Glia

Research report
Inhibition of p38 mitogen-activated protein kinase activation in the rostral anterior cingulate cortex attenuates pain-related negative emotion in rats